The NMDA receptor subtype is known to have a fundamentally important role in CNS function. Ongoing work from many laboratories has established the involvement of NMDA receptors in multiple aspects of brain development, synaptic plasticity associated with long-term potentiation, and pathology related to glutamate-mediated excitotoxicity. In particular, neuropathological mechanisms mediated by NMDA receptors have recently been implicated in neurological disorders including ischemic stroke, kindling epileptogenesis, and schizophrenia.
The NMDA receptor is composed of two different types of subunits, the NR1 and the NR2. The NR1 subunit is ubiquitously expressed throughout the CNS, particularly in the cerebral cortex, hippocampus, and olfactory bulb. While homomeric NMDA receptors composed of NR1 subunits are activated by NMDA, heteromeric receptors composed of both NR1 and NR2 subunits exhibit greater responses to NMDA.
Alternative splicing of three exons, xcex1, xcex2, and xcex3 (also referred to in the art as N1, C1, and C2, respectively) generates eight isoforms of the mRNA encoding the NR1 protein. A schematic of the NR1 splice variants is shown in FIG. 1 in which the presence or absence of any of the three alternatively spliced exons is designated by a subscript within the name. FIG. 2 lists the eight splice variants and the alternatively spliced exons present in each variant. Exons, xcex1, xcex2, and xcex3, code for 21, 37, and 38 amino acid sequences, respectively. The xcex1 exon, which corresponds to exon 5, is located in the extracellular amino-terminal portion of the receptor, whereas the xcex2 and xcex3 exons, corresponding to exons 21 and 22, respectively, are located at the carboxy-terminal domain. Interestingly, a stop codon is contained within the xcex3 exon. Its removal by alternative splicing leads to the inclusion of a new 22 amino acid sequence.
The inclusion of any one or a combination of the three exons imparts differential pharmacological properties to the NMDA receptor. For example, inclusion of the N1 exon leads to a decrease in agonist affinity, but an increase in current amplitude. A number of consensus PKC phosphorylation sites have been identified within the C1 exon; however, a functional role for these sites has not been fully elucidated. Other differences imparted by alternative splicing include sensitivity to potentiation by spermine, Zn2+, and activators of PKC.
An examination of the levels of NR1 splice variants in the developing rat brain has revealed regional differences in the pattern of expression. This divergent pattern in the expression of the NR1 splice variants may provide a degree of functional diversity in NMDA receptor function that underlies the regional heterogeneity in certain NMDA receptor-dependent processes, including synaptic consolidation, potentiation, and plasticity.
The four subtypes of the NR2 subunit: NR2A, NR2B, NR2C, and NR2D were identified through molecular cloning studies. Studies indicate that the inclusion of different NR2 subtypes within the heteromeric NMDA receptor alters the pharmacological properties of NMDA receptor function providing another level of functional diversity to the receptor. For example, differences in Ca2+ permeability, sensitivity to Mg2+ block, glycine potentiation, and offset kinetics can be attributed to the presence of specific NR2 subunits in the heteromeric receptor.
The NR2 subunits have about 50% sequence homology between members and have approximately 15% sequence homology with the NR1 subunits. They are incapable of forming functional NMDA-activiated channels following homomeric expression, but rather serve to potentiate the NMDA response and modify pharmacological properties when coexpressed with NR1 subunits. Recent work has shown that the NR2A and NR2B subunits are tyrosine-phosphorylated, whereas the NR1 subunits are not.
In situ hybridization experiments have shown that the NR2 subunits exhibit a region-specific and temporal-specific pattern of expression. For example, the NR2B subunit is mainly expressed in the rat forebrain, whereas the NR2C subunit is found predominantly in the cerebellum. Differences in the expression of the NR2 subunits may be responsible for imparting functional diversity to NMDA receptor function from one cell type to the next.
The present invention relates to a method for identifying a subunit specific modulator of the N-methyl-D-aspartate (NMDA) receptor. The method involves providing a plurality of NMDA receptors which differ in their subunit identity. The receptors are contacted with a neurotransmitter recognition site ligand in the presence and absence of a candidate modulator. Receptor activity is then assayed, with an increase or decrease in activity in at least one, but not all members of the plurality of NMDA receptors, in the presence but not the absence of a candidate modulator, being an indication that the candidate modulator is a subunit specific modulator. The subunit identity of the subset of the NMDA receptors to determine the subunit specificity of the candidate modulator. Various combinations of NMDA receptor subunits are provided.